Our broad objective is to investigate the biochemical basis of hypermutation, which when properly regulated is essential for generating antibody diversity, but when unregulated leads to human disease. Our focused objective is to investigate a family of mutator enzymes, the APOBEC dC deaminases that convert C to U on single-stranded DNA resulting in genomic mutations. On the positive side of the hypermutagenic coin, activation-induced deoxycytidine deaminase (AID) deaminates C to U during transcription of immunoglobulin genes to initiate somatic hypermutation (SHM) and class-switch recombination (CSR) required to produce high affinity antibodies. Apo 3G and Apo 3A play instrumental roles in restricting infection of the AIDS virus (HIV-1) by deaminating C on retroviral cDNA. However, on the negative side of the hypermutagenic coin, it's recently been shown that AID, Apo3A, and Apo3B are involved in numerous types of cancer when acting inadvertently, i.e., off-target on genomic DNA. From a biological perspective, an understanding of the biochemical properties of the APOBEC enzymes is essential to grasp the programmed and non-programed hypermutation roles for these enzymes. In the previous grant period, we've developed biochemical and mathematical methods to establish deamination/mutation signatures for the APOBECs. We now propose to identify individual mutagenic contributions of AID, Apo3A, Apo3B to specific cancer hypermutation clusters (kataegis) identified in cancer genomes by generating deamination signatures in vitro, with each enzyme acting on single-stranded and transcribed double-stranded DNA kataegis regions (Aim 1). We've recently developed an assay using nuclear extracts from Ramos B cells to study AID-catalyzed dC deamination during transcription by human RNA polymerase II; this is the first biochemical analysis of AID acting in conjunction with RNAP II. The human transcription system will allow us to determine the roles of transcriptional factors that target AID to either moving or stalled transcription bubbles, and to identify new factors that interact with AID and/or human RNAP II (Aim 2). In Aim 3, total internal reflection fluorescence (TIRF) microscopy will allow us to visualize the dynamics of AID, Apo3A, and Apo3B moving on IgV, IgS and cancer kataegis regions in single-stranded and transcribed dsDNA at single molecule (sm) resolution, in real-time. The recent recognition that the inadvertent action of AID, Apo3A and Apo3B can have important cancer etiologic consequences is likely to represent just the tip of the iceberg. Our combined biochemical-mathematical strategy to identify characteristic spatial mutational signatures for AID, Apo3A and Apo3B is poised to play a key translational role in ongoing studies to identify the source of each of these dC deaminases in initiating human cancer-associated mutations; more importantly our approach can serve as a benchmark for future studies to establish the involvement of any APOBEC family member in initiating cancer.